PROJECT SUMMARY/ABSTRACT Non-alcoholic fatty liver disease (NAFLD), typically associated with overnutrition and obesity, is one of the most common liver diseases, both in the U.S. and worldwide, with a prevalence of 20-30%. During obesity and NAFLD, lipotoxic injuries to hepatocytes can induce the formation of protein inclusions consisting of p62/SQSTM1 and ubiquitinated proteins. These inclusions can contribute to the pathological progression of NAFLD to more severe forms of liver diseases, such as non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, we currently do not have any mechanistic understanding of how lipotoxicity stimulates protein inclusion formation during obesity and NASH, and how this pathway plays a pathogenetic role during NASH-associated HCC progression. Our goal in this project is to mechanistically understand how protein inclusions are formed during obesity and how they contribute to the progression of NASH-HCC pathologies. Our central hypothesis is that TBK1, a protein kinase activated upon lipotoxic insults during obesity, phosphorylates p62 and that this phosphorylation is an important pathogenetic event that contributes to protein inclusion body formation and promoting HCC development. The scientific premise supporting our research, provided by recently published findings and our own preliminary data, includes (1) activation of hepatic TBK1 during obesity, (2) critical role of p62 in NASH-to-HCC progression, (3) genetic requirement of TBK1 in obesity-induced p62 phosphorylation and accumulation, and (4) strong impact of obesity and lipotoxicity on HCC progression. Based on these findings, we further propose to investigate how TBK1 is activated during obesity (Aim 1) and whether and how the TBK1-mediated p62 control contributes to progression of NASH to HCC (Aim 2). With respect to expected outcomes, the work proposed in aims 1 and 2 will result in delineation of a signaling pathway that mediates formation of insoluble protein inclusions upon lipotoxic insults and show its pathological relevance in NASH-associated HCC. Such results are expected to have an important positive impact, because this new knowledge may lead to development of innovative therapeutic rationale for NASH-associated HCC, by pharmacological inhibition of the identified pathway components.